The long term goal of this project is to elucidate cellular and molecular mechanisms of development. This is basic research that will contribute to the understanding and eventual amelioration of genetic and environmentally induced developmental disorders and to the training of future biomedical health researchers. The rationale for the proposed research is as follows: It is increasingly clear that, as with many metabolic and structural proteins, many developmental regulatory proteins also show a high degree of evolutionary conservation, so that homologs of these genes, identified mainly by genetic techniques in Drosophila, can be identified in vertebrate and other invertebrates. An important part of understanding how these genes work is to understand how their embryonic expression and developmental effects on individual cells are governed by interactions with other cells. But in Drosophila and vertebrate embryos it is difficult to study development at the level of single cells because of technical difficulties such as the small size and large numbers of cells, difficulty in culturing the embryos, and (in Drosophila) the fact that early development involves a syncytial blastoderm stage, so that many early events occur in an environment in which individual cells cannot be identified or examined. Fortunately, we can carry out such cell-level studies of developmental regulatory genes in leech with relative ease, because in leech embryos, individually identified cells are accessible to experimental manipulations both in the embryo and in the adult nervous system. The specific aims of this project are as follows: l) to continue characterizing the normal expression of the leech gene ht-en (a homolog of the Drosophila segmentation gene engrailed; the question of immediate interest is whether expression of engrailed-class genes is associated with particular cell phenotypes in the embryo and/or in the nervous system); 2) to look for cell fate changes after selective ablation of cells that express ht-en; 3) to look for cell fate changes after specific mis-expression of ht-en; 4) to characterize the expression of leech homologs of other segmentation genes, such as cubitus m.interruptus-Dominant and to test for the presence of interactions among the cells expressing these genes and the cells expressing ht-en.